Our overall research initiative is focused on understanding the molecular basis of aberrant DNA methylation and its role in human carcinogenesis. Gene silencing associated with the aberrant methylation of promoter region CpG islands is a common epigenetic alteration that contributes to the inactivation of tumor suppressor and other genes in human cancers. At present, there is little known about how or why particular genes succumb to this aberrant event, and the mechanisms in which an active gene progresses to a methylated and inactive state are not well understood. Emerging evidence linking DNA methylation with posttranslational modification of histones suggests that the local chromatin architecture may be a critical determinant of normal, and abnormal, DNA methylation patterning. We propose that the aberrant methylation arises from a failure in cis of a mechanism that blocks or actively opposes the spread of heterochromatin. The goal of this renewal application is to further explore the idea that CpG islands define a distinct domain at the level of chromatin, and that the integrity of this domain prevents aberrant methylation in normal cells. Our studies will focus on a novel gene identified in our laboratory, TMS1, that is silenced by epigenetic means in human breast and other cancers. Specifically, we plan to determine whether DNAsel hypersentitive sites identified at the boundary between the unmethylated CpG island and methylated flanking DNA act in cis to protect the CpG island from de novo methylation and the influences of surrounding chromatin. Secondly, we will characterize a novel methylation-sensitive complex that binds to the TMS1 CpG island in its unmethylated state. Finally, we will test the hypothesis that aberrant methylation is prevented by the maintenance of a euchromatic mark over CpG island chromatin. A better understanding of the mechanisms underlying the genesis and consequences of aberrant DNA methylation during carcinogenesis will provide for the future development of novel treatment strategies aimed at preventing or reversing the silencing process.